The present invention relates to a thermally stable enzyme composition and a method of manufacturing the same, and more particularly, to an enzyme composition thermally stable in an aqueous solution and a method of manufacturing the same.
An enzyme can effect a catalytic reaction under milder conditions and exhibits a high substrate specificity, compared with a chemical reaction. Therefore, an enzyme is widely used in the fields of food industry, chemical industry, pharmaceutical industry, etc.
However, the enzyme is generally unstable. Particularly, the enzyme stability is low in a solution. It should be noted that a thermal denaturation begins to take place in many lipid-decomposing enzymes if these enzymes are exposed to temperatures exceeding about 35xc2x0 C., making it difficult to utilize an enzyme in a reaction system using a substrate having a high melting point. Also, since an enzyme is thermally unstable, a freeze-drying method that is less likely to be affected thermally is utilized mainly for concentrating and preparing an enzyme powder. However, the freeze-drying method requires a tremendous facility investment for the mass production, resulting in a high running cost such as a utility cost.
For improving the thermal stability of the enzyme within an aqueous solution, it has been proposed to add a stabilizer to the enzyme. For example, it has been studied to add an amino acid such as albumin, casein, or sodium glutamate; a reducing agent such as protein, mercapto ethanol, or cysteine; a polyol such as glycerol, sucrose, or sorbitol; or a water-soluble high molecular weight substance such as dextran to an enzyme solution. Also proposed are a method of modifying an enzyme surface with, for example, an emulsifier or another amphoteric substance as disclosed in Japanese Patent Disclosure (Kokai) No. 6-113847, a method of modifying an enzyme surface with an oil-soluble substance having an isoprenoid structure as disclosed in Japanese Patent Disclosure No. 6-269285, and a method of stabilizing an enzyme by dissolving a phospholipid in an alcoholic solvent, followed by mixing the resultant solution with an enzyme and subsequently drying the mixture (Japanese Patent Disclosure No. 1-27719).
However, the lipid-decomposing enzyme obtained by the conventional methods described above is stable only under temperatures of about 37 to 60xc2x0 C. and, thus, is relatively unstable under heat, making it relatively difficult to use the enzyme for an enzyme synthesis, etc. using a substrate having a high melting point. Also, in the method of preparing an enzyme powder by a spraying method, which is a typical drying method, the enzyme is concentrated under heat and, then, dried within a hot gaseous stream. Therefore, the enzyme activity is lowered.
An object of the present invention is to provide a thermally stable enzyme composition exhibiting an improved heat resistance even within an aqueous solution and capable of stably performing an enzyme reaction under high temperatures.
Another object is to provide a method of preparing an enzyme composition that permits stably preparing an enzyme powder while preventing the enzyme activity from being lowered by the heat in the enzyme powder preparation even by a drying method under heat.
These objects can be achieved according to the present invention by adding a heat stabilizer comprising a combination of an oil-soluble vitamin and a phospholipid to an enzyme solution. The heat stability of the enzyme is significantly improved by adding a heat stabilizer including an oil-soluble vitamin and a phospholipid.
Thus, the present invention provides an enzyme composition comprising an enzyme and a heat stabilizer including a phospholipid and an oil-soluble vitamin.
The present invention also provides a method of preparing a thermally stable enzyme, comprising the step of drying an enzyme solution containing a phospholipid and an oil-soluble vitamin to obtain an enzyme powder. The drying should preferably be performed by a spray drying.
In a preferred embodiment of the present invention, the enzyme composition of the invention contains 0.01 to 200% by weight of a phospholipid and 0.01 to 100% by weight of an oil-soluble vitamin, based on the enzyme weight.
The enzyme used in the present invention should desirably be a lipid-decomposing enzyme. Also, it is desirable to use an oil-soluble vitamin selected from the group consisting of tocopherol, tocotrienol, retinol, calciferol, phylloquinone, and ubiquinone.
The present invention provides a method of preparing an enzyme, in which a phospholipid and an oil-soluble vitamin are added to an enzyme solution to improve the thermal stability of the enzyme and which permits suppressing the deactivation of the enzyme by heat in a powder preparation by a drying method under heating to make it possible to prepare an enzyme powder.
The enzyme used in the present invention, which is not particularly limited, includes preferably a lipid-decomposing enzyme, protease, and sugar-decomposing enzyme. It is particularly desirable to use a lipid-decomposing enzyme. In addition to the enzyme formulations available on the market, the enzyme can be used in the present invention in the form of a microorganism culture solution, a plant extraction liquid and an animal cell extraction liquid. Further, a culture solution, a concentrated extraction liquid and a desalted concentrated solution can also be used as the enzyme in the present invention.
The lipid-decomposing enzyme used in the present invention includes lipases, phospholipases, and esterases. The lipases include, for example, lipoprotein lipase, monoacyl glycerollipase, diacyl glycerollipase, triacyl glycerollipase, and galactolipase. The phospholipases include, for example, lyso phospolipase, and phospholipases A1, A2, B, C and D. Further, the esterases include, for example, choline esterase, cholesterol esterase, pectin esterase, tropine esterase, acetylcholine esterase, acetyl esterase, carboxy esteradse, and aryl esterase.
The sugar-decomposing enzymes used in the present invention include, for example, amylase, glucosidase, cellulase, xylanase, dextranase, chitinase, lysozyme, galactosidase, mannosidase, glucuronidase, hyaluronidase and pectin lyase.
The protease used in the present invention includes endopectidases and exopeptidases. The endopectidases include, for example, acid proteinase, serine protease, cysteine protease, asparagic acid protease, thiol protease, and carboxy protease. On the other hand, the exopetidases include, for example, dipeptidylamino peptidase and dipeptidylcarboxy peptidase.
The microorganisms producing the enzymes used in the present invention, which are not particularly limited, can be selected from bacteria, yeasts, filamentous viruses, and actinomycetes, and include, for example, Psudomonas species, Alcaligenes species, Arthrobacter species, Staphylococcus species, Torulopsis species, Escherichia species, Micotorula species, Propionibacterum species, Chromobacterum species, Xanthomonas species, Lactobacillus species, Clostridium species, Candida species, Geotrichum species, Sacchromycopsis species, Nocardia species, Fuzarium species, Aspergillus species, Penicillium species, Mucor species, Rhisopus species, Phycomycese species, Puccinia species, Bacillus species and Streptmycese species.
A culture medium containing soybean powder, peptone, corn steep liquor, K2HPO4, (NH4)2SO4, MgSO4.7H2O, etc. can be used for growing the microorganisms given above. It is. desirable for the culture medium to contain soybean powder in an amount of 0.1 to 20% by weight, preferably 1.0 to 10% by weight. Peptone should desirably be contained in an amount of 0.1 to 30% by weight, preferably 0.5 to 10% by weight. Corn steep liquor should desirably be contained in an amount of 0.1 to 30% by weight, preferably 0.5 to 10% by weight. K2HPO4 should desirably be contained in an amount of 0.01 to 20% by weight, preferably 0.1 to 5% by weight. (NH4)2SO4 should desirably be contained in an amount of 0.01 to 20% by weight, preferably 0.05 to 5% by weight. Further, MgSO4.7H2O should desirably be contained in an amount of 0.01 to 20% by weight, preferably 0.05 to 5% by weight. Other culture media can also be used, as exemplified in the Examples described herein later. It is desirable to culture the microorganisms under the culturing temperature of 10 to 40xc2x0 C., preferably 20 to 35xc2x0 C., an air circulation rate of 0.1 to 2.0 VVM, preferably 0.1 to 1.5 VVM, a stirring rotation speed of 100 to 800 rpm, preferably 200 to 400 rpm, and a pH of 3.0 to 10.0, preferably 4.0 to 9.5.
The method of extracting the enzymes from the culture is not particularly limited in the present invention, though it is desirable to remove the bacterial cells by means of centrifugal separation, membrane filtration, etc. in the case of exoenzymes. It is desirable to carry out the centrifugal separation under a centrifugal force of 200 to 20,000xc3x97g. It is desirable to carry out the membrane filtration while controlling the pressure at 3.0 kg/m2 or less by using an MF membrane or a filter press. In the case of endoenzymes, it is desirable to disintegrate the cells by using a homogenizer, a Waring blender, an ultrasonic disintegrator, a French press, a ball mill, etc. and to remove the cell residue by centrifugal separation, a membrane filtration, etc. The stirring rotating speed of the homogenizer should be 500 to 30,000 rpm, preferably 1,000 to 15,000 rpm. The Waring blender should be scanned at a rate of 500 to 5,000 rpm, preferably 1,000 to 10,000 rpm, and the stirring should be performed for 0.5 to 10 minutes, preferably 1 to 5 minutes. The ultrasonic disintegrator should be scanned at 1 to 50 KHz, preferably 10 to 20 KHz. Further, glass balls having a diameter of about 0.1 to 0.5 mm should desirably be used in the ball mill.
The phospholipid that is used as a component of the heat stabilizer of the invention is not particularly limited in the present invention, and includes, for example, a phospholipid derived from plant seeds, a phospholipid derived from animals, and a phospholipid prepared by an enzyme synthesis or a chemical synthesis. The plant seeds include, for example, rapeseeds, safflower seeds, soybean, corn, sesame seeds, and cotton seeds. The animal-derived phospholipid includes, for example, phospholipids derived from egg yolk and cattle brain. Further, the phospholipid prepared by enzyme synthesis or chemical synthesis, which is not particularly limited in the present invention, includes, for example, high purity products of PS (phosphatidyl serine), PI (phosphatidyl inositol), PA (phosphatidic acid), LPE (lysophosphatidyl ethanolamine), and LPC (lysophosphatidyl choline) that are produced by a base exchange reaction or hydrolytic reaction using phospholipase or a chemical synthesis. The kinds of the phospholipids, which are not particularly limited in the present invention, include, for example, PC (phosphatidyl choline), PE (phosphatidyl ethanol), PA, PI, and their lyso forms.
The oil-soluble vitamin that is used as another component of the heat stabilizer, which is not particularly limited in the present invention, should desirably be xcex1-, xcex2-, xcex3-tocopherols, tocotrienol, retinol, calciferol, menaquinone, menadione, phylloquinone, ubiquinone and a mixture thereof.
For preparing a thermally stable enzyme composition of the present invention, an aqueous solution of an object enzyme is prepared, followed by adding a phospholipid and an oil-soluble vitamin to the aqueous solution for sufficiently mixing these components in the solution. Then, the enzyme aqueous solution having the heat stabilizer added thereto can be concentrated and converted into a powder.
An aqueous solution of the enzyme can be prepared by dissolving the dry enzyme powder in water. Alternatively, a supernatant of the enzyme culture solution or the extraction liquid itself can be used as the enzyme aqueous solution.
The enzyme concentration of the aqueous solution, which is not particularly limited in the present invention, should desirably be 0.01 to 90% by weight in the case of using a supernatant of the culture or the extraction liquid and 0.01 to 90% by weight in the case of using a dry enzyme powder.
To be more specific, in order to modify the surface of the enzyme with the heat stabilizer of the present invention, a phospholipid may be added in an amount of preferably 0.01 to 200% by weight, more preferably 1 to 8% by weight, of the enzyme weight to the aqueous solution of the object enzyme while maintaining the solution temperature at 0 to 25xc2x0 C., preferably 0 to 5xc2x0 C., and while stirring the solution with a homogenizer at 300 to 10,000 rpm for 1 to 30 minutes, preferably 1 to 5 minutes. Then, an oil-soluble vitamin may be added in an amount of preferably 0.01 to 100% by weight, more preferably 0.01 to 20% by weight, of the enzyme weight, to the solution while stirring the solution with a homogenizer at a rate of 100 to 8,000 rpm, preferably 1,000 to 6,000 rpm, for 1 to 20 minutes, preferably 1 to 5 minutes. The maximum yields of the concentrate and the powder depend on the concentration and kind of the enzyme, phospholipid and oil-soluble vitamin, making it possible to determine appropriately the concentrations of these components from within the ranges given above.
The enzyme composition of the present invention can be concentrated by a suitable concentrating method. For example, the concentration can be achieved by means of an evaporator, a flash evaporator, ultrafiltration (UF), membrane concentration, MF membrane concentration, salting-out with inorganic salts, precipitation using a solvent, adsorption using an ion-exchange cellulose or the like, and hygroscopic method using a hygroscopic gel. Particularly, a UF membrane concentration and concentration using an evaporator are preferably used. Concerning the module for the UF membrane concentration, it is desirable to use a plain membrane or a hollow fiber membrane having a fraction molecular weight of 3,000 to 100,000, preferably 6,000 to 50,000. It is also desirable to use a polyacrylonitrile material or a polysulfone material for forming the UF membrane. The concentration using an evaporator should desirably be performed under a heated temperature of 90xc2x0 C. or less and a reduced pressure of 40 cmHg or less, more desirably under a heated temperature of 80xc2x0 C. or less and a reduced pressure of 60 cmHg or less.
The enzyme composition of the present invention can be converted into a powder satisfactorily by any of drying under a reduced pressure, a freeze drying and a spray drying. It is desirable to employ a freeze drying or a spray drying in view of the activity recovery of the enzyme. The spray drying is particularly desirable if the production efficiency is also taken into account. The spray dryer includes, for example, a nozzle counter current type, a disc counter current type, a nozzle parallel flow type, and a disc parallel flow type. Preferably, it is desirable to use a disc parallel flow type spray dryer. For operating the spray dryer, it is desirable to control the rotating speed of the atomizer at 4,000 to 20,000 rpm, the inlet temperature at 100 to 200xc2x0 C. and the outlet temperature at 40 to 100xc2x0 C.